Interaction between Mcm and Cdt1. A–E, purified proteins, singly or in combination, were fractionated by centrifugation at 36,000 rpm for 17 h on 15–35% glycerol gradient. Each fraction was run on 4–20% SDS-PAGE, and proteins were stained with silver. The buffer used in the gradient was 50 mm sodium acetate, 50 mm Hepes-Na, pH 7.5, 0.5 mm EDTA, 1 mm DTT, 0.1 mm phenylmethylsulfonyl fluoride, 0.01% Triton X-100, 2 mm Mg-acetate, and 1 mm ATP. A, Cdt1; B, Mcm4/6/7; C, Mcm4/6/7+Cdt1; D, Mcm2/3/4/5/6/7+Cdt1; E, Mcm2/4/6/7+Cdt1. The positions of molecular weight markers are indicated along with their molecular sizes. BC, samples applied. F, purified Mcm3/5 (m3/5), Mcm4/6 (m4/6), and Mcm2 (m2) proteins were mixed with T7-tagged Cdt1 purifiedfromE. coli, and immunoprecipitation was performed using T7-antibody beads. Unbound (U) and bound (B) fractions were analyzed on 7.5% SDS-PAGE, and proteins were visualized by silver staining.

Effect of geminin on helicase and DNA binding activities of Mcm4/6/7 in the presence of Cdt1. The helicase (A) and gel shift (B) assays were carried out as described under “Experimental Procedures.” Amounts of proteins used are shown above each panel. Geminin WT and Δ represent, respectively, the wild-type protein and a mutant protein lacking the Cdt1-interacting domain. The wild-type geminin but not the mutant strongly inhibited the helicase activity of Mcm4/6/7 in the presence of Cdt1 protein and disrupted the Cdt1-Mcm4/6/7-DNA complex, regenerating the Mcm4/6/7-DNA complex. * indicates weak shifted bands generated by the wild-type geminin preparation. The band indicated by ** appears only when Tris and Hepes buffers derived from the reaction mix and protein preparations, respectively, are mixed, and could represent the substrate DNA adopting a different conformation.

Cdt1 forms high molecular weight complexes with Mcm4/6/7 in the presence of ATP. Purified Mcm4/6/7 with or without Cdt1 protein (purified from insect cells; A and B, wild-type; C and D, KKAA mutant) were fractionated by glycerol gradient (15–30%). The buffer used in the gradient was 50 mm sodium acetate, 50 mm Hepes-Na, pH 7.5, 0.5 mm EDTA, 1 mm DTT, and 0.01% Triton X-100, and 2 mm Mg-acetate, with (A, C, E, and G) or without (B, D, and F) 1 mm ATP. Centrifugation was at 38,000 rpm for 17 h. Each fraction was run on 4–20% SDS-PAGE, and proteins were stained with silver. The positions of molecular weight markers are indicated along with their molecular sizes. H, Cdt1 KKAA mutant protein (purified from insect cells) fractionated by glycerol gradient (15–30%) at 45,000 rpm for 20 h.

Cdt1 stimulates DNA binding and helicase activities of Mcm4/6/7. DNA binding (A and C) and helicase (B and D) activities were examined with a constant amount of Mcm4/6/7 (60 ng/4.4 nm (A and B) or 30 ng/2.2 nm (C and D)) and various amounts of Cdt1 protein purified from insect cells (A and B) or from E. coli (C and D). A and C, DNA binding activity was measured in gel shift assays using ssDNA (20 fmol) as substrates. Complexes were separated on a 5% native polyacrylamide gel. The bands indicated by brackets in lanes 3– 8 (A) or lanes 3–7 (C) represent the Cdt1-Mcm4/6/7-DNA complex. B and D, DNA helicase assays were conducted as in A and C, respectively, except that the partial heteroduplex substrate (10 fmol) was used. Because the purified Cdt1 protein from the insect cell contained 2 mm ATP, complement buffer was added to maintain the final reaction mix as follows: 50 mm Tris-HCl, pH 7.6, 60 mm sodium acetate, 6% glycerol, 0.3 mm EDTA, 0.6 mm DTT, 20 mm 2-mercaptoethanol, 0.006% Triton X-100, 0.25 mg/ml bovine serum albumin, 11.2 mm MgOAc, and 11.2 mm ATP. Half of the reaction was incubated at 30 °C for the gel-shift assay and the remainder was incubated at 37 °C for the DNA helicase assay (A and B). In A, the extent of DNA binding with Mcm or Tag alone was taken as 1, and the relative efficiency of DNA binding in the presence of various amounts of Cdt1 protein is presented for each helicase. In B, the level of the displaced oligonucleotide was quantified, and that with Mcm or Tag alone was taken as 1. The relative level of DNA unwinding in the presence of various amounts of Cdt1 protein is presented for each helicase. D, heat-denatured substrates.

Effect of ATP concentration on DNA binding and helicase activities of Mcm4/6/7 in the absence and presence of Cdt1. DNA helicase (A) and DNA binding (B) activities of Mcm4/6/7 (40 ng) in the absence (lanes 2–6) or presence (lanes 7–11) of Cdt1 (30 ng) were examined with increasing concentrations of ATP. The level of DNA helicase activity, calculated as percent oligonucleotide displaced, is presented at various ATP concentrations. The stimulation of helicase activity by Cdt1 was more prominent at 10–13 mm ATP.

Biochemical characterization of Cdt1 (KKAA) mutant protein. A, schematic drawing of domain structures of Cdt1, and the mutagenized amino acids. B, using the identical amount of wild-type or KKAA Cdt1 protein, the effect on DNA binding (lower panel) and helicase (upper panel) activities of Mcm4/6/7 were examined. The proteins used were; 30 ng of Mcm4/6/7 and 10, 20, 40, and 80 ng of Cdt1. C, kinetics of helicase reactions using 30 ng of Mcm4/6/7 and 15 ng of Cdt1 (wild type or KKAA). D, pull-down assay was carried out with 50 ng of Cdt1 (wild type or KKAA) and 50 pmol of biotinylated oligonucleotide in the presence or absence of ATP. The bound (left half) and unbound (right half) proteins are shown. * indicates nonspecific bands. E, purified Mcm4/6/7 (200 ng) was mixed with the same amount (200 ng) of wild-type or KKAA T7-tagged Cdt1, and immunoprecipitation experiments were performed using T7-antibody beads as described under “Experimental Procedures,” except that beads were further washed twice with T7-wash buffer (4.29 mm Na₂HPO₄, 1.47 mm KH₂PO₄, 2.7 mm KCl, 137 mm NaCl, and 0.1% Tween-20, pH 7.3). F, purified geminin (200 ng) was mixed with same amount (200 ng) of wild-type or KKAA T7-tagged Cdt1, and immunoprecipitation experiments were performed using T7-antibody beads as described under “Experimental Procedures.” In D, E, and F, the samples were run on 4–20% SDS-PAGE, and the proteins were detected by silver staining. In E and F: I, input; U, unbound; B, bound. Cdt1 was overexpressed and purified from insect cells (B and C) or from E. coli (D, E, and F). The KKAA mutant did not promote the high molecular weight complex formation with Mcm at a higher concentration and did not stimulate the helicase activity of Mcm4/6/7. The interaction of the mutant Cdt1 with Mcm was significantly reduced, although it interacted with DNA and with geminin as efficiently as wild-type.